Modulation of mesenchymal cells via iga-receptors

ABSTRACT

IgA receptors, including a polymeric immunoglobulin receptor (pIgR) and a FcαR, have been found on smooth muscle cells, synovial fibroblast cells and on both synovial and endothelial cells in synovial tissues from patients with arthritis. Incubation of smooth muscle cells or tissue with pIgA increases cytosolic calcium and alters the contractile state. Incubation of synovial cells with IgA modulates the inflammatory responses of these cells. The invention relates to methods of modulating calcium signalling and/or contractility of mesenchymal cells, as well as modulating (preferably inhibiting) the inflammatory responses of mesenchymal cells, methods of treating inflammatory conditions (such as asthma and arthritis), methods of drug delivery to mesenchymal cells and methods of detecting conditions associated with IgA receptors on mesenchymal cells.

FIELD OF THE INVENTION

The invention relates to methods of modulating mesenchymal cell biology,including cytosolic calcium signalling in and inflammatory responses ofmesenchymal cells, methods of treating arthritis and asthma and methodsof drug delivery to mesenchymal cells, as well as methods to diagnoseIgA-receptor-mediated mesenchymal inflammation.

BACKGROUND OF THE INVENTION

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a common chronic inflammatory andautoimmune disorder of unknown etiology that attacks adults and children(Choy and Panayi, 2001). Patients with RA have a poor long-termprognosis, with 80% becoming disabled after 20 years (Scott et al,1987). Current treatments do not improve this prognosis. The prevalenceof RA is about 1.3% and has been steadily rising. Insights into the cellbiology of this disorder will go far to developing improved treatmentsthat prevent disability and improve long-term prognosis.

RA is characterized by synovial inflammation, proliferation andprogressive joint destruction (reviewed in Jenkins et al, 2002). Theimmune reaction begins in the synovial lining of the joint, withlymphocytes playing a significant role in acute disease and a lesserrole in chronic disease. The earliest pathologic changes in the diseaseare microvascular injury and increased vascular permeability,accompanied by an influx of inflammatory cells (CD4 lymphocytes,neutrophils, and plasma cells) in the perivascular space. Cytokines,lymphokines, and chemokines are released. TNFα, IL-1 and IL-6 are thekey cytokines that drive inflammation in RA. Patients develop swelling,pain, and joint stiffness with the onset of vascular injury andangiogenesis in the synovial membrane. Synovial proliferation and theevolving inflammation exacerbate these symptoms and progressively limitjoint motion. Neutrophils accumulate in the synovial fluid in responseto local production of IL-8. B lymphocytes mature into plasma cellswhich locally produce rheumatoid factor and other antibodies thatfurther aggravate the inflammation. Immune complexes activate thecomplement system, releasing chemokines and increasing vascularpermeability. Immune complexes also promote phagocytosis, leading togreater lysosomal enzyme release and the digestion of collagen,cartilage matrix, and elastic tissues. The release of oxygen freeradicals injures cells, which release phospholipids that fuel thearachidonic acid cascade and exacerbate the local inflammatory response.Proliferating synovium forms an invasive pannus, eroding throughcartilage and subchondral bone. Of the two types of synoviocytes, type Amonocyte-like and type B fibroblast-like, the type B fibroblast-likecells stimulate the cartilage and bone destruction of chronic disease.Chondrocytes release their own proteases and collagenases, and furthercontribute to this self-perpetuating local immune response.

The initial inciting factor and the precise mechanism of these complexcellular interactions remain unknown. However, it is clear that RA ischaracterized by increased activity of the pro-inflammatorytranscription factor, NFκB, in synovial fibroblasts. NFκB stimulatesproduction of cytokines and adhesion molecules, including TNF-α, IL-1β,IL-6, IL-8 and ICAM-1.

Patients with RA may also develop systemic vasculitis, neurologic,pulmonary, cardiac and/or liver abnormalities. The number and severityof the extra-articular features vary with the duration and severity ofdisease. Extra-articular complications are seen in patients with hightiters of rheumatoid factor (RF). RF is an immunoglobulin that bindsother immunoglobulins at their Fc components, forming immune complexes.RF may consist of IgM, IgA, IgE and/or IgG isotypes, and may be found inseveral other diseases including Sjogren's syndrome, subacute bacterialendocarditis, mixed cryoglobulinemia, systemic lupus erythematosis,scleroderma, sarcoidosis, idiopathic pulmonary fibrosis andmalignancies. However, the combined elevation of IgM-RF and IgA-RF ishighly specific for RA and is very rarely found in rheumatic diseasesother than RA (Jonsson et al, 1998). In a cross-sectional study, themajority (74%) of RA patients had elevations of 2-3 RF isotypes, and 67%had the combined elevation of IgA and IgM (Jonsson and Valdimarsson,1992). Of those patients with RA, 65% are positive for IgA-RF and 92%are positive for IgM-RF (Gioud-Paquet et al, 1987).

IgA-RF can occur in serum and synovial fluid, and is predominantlypolymeric (Otten et al, 1991; Schrohenloher et al, 1986). Severalstudies have reported significant clinical implications to IgA-RF in RA.RA patients with a predominant increase in IgA-RF have more erosivedisease (Jorgensen et al, 1996). IgA-RF Is associated withextra-articular manifestations of RA (Jonsson et al, 1995; Pai et al,1998). Detection of IgA-RF early in disease predicts poorer prognosiswith a more rapidly progressive course (Teitsson et al, 1984; Pai et al,1998; Houssien et al, 1997).

Asthma

Asthma is a chronic inflammatory airway disease with symptoms ofwheezing, cough, shortness of breath and chest tightness. These symptomsvary from person to person and occur with no set pattern. The cause ofasthma is not known and there is no cure. Asthmatics respond diverselyto available treatments. Asthma triggers also vary from person toperson. Viral respiratory infections play an important role in asthma,triggering an asthma attack and sensitizing patients to other triggers,such as dust mites, animals, molds, pollens and air pollutants (Messageand Johnston, 2002; Tuffaha et al, 2000). Other symptom triggers includesmoke, exercise, cold air, increased humidity, strong-smellingsubstances (e.g. perfume, cigarette smoke, paint fumes, dusts), sinusinfection, gastric aspiration or gastroesophageal reflux, and certainfood additives. Not all asthmatics are alike in their responses to thesedifferent stimuli. Some people react immediately to a stimulus; othershave delayed bronchial constriction. Furthermore, different patientsrespond differently to currently available medications for asthma.Regardless of the heterogeneity of asthma, bronchial hyper-reactivity isits hallmark and consists of smooth muscle contraction along thebronchial tree.

A number of studies indicate a major role for alterations in the smoothmuscle. Airway smooth muscle (ASM) cells exhibit a contractile phenotypeand a proliferative-synthetic phenotype, capable of producingproinflammatory cytokines, chemokines and growth factors (Halayko et al,1996; Halayko et al, 1999; Schmidt and Rabe, 2000). It is now beingsuggested that the ASM itself can contribute directly to the persistenceof inflammation and airway remodeling that occurs in asthma. Thefeatures of this airway remodeling include epithelial damage, depositionof extracellular matrix proteins throughout the airways, goblet cellmetaplasia, and smooth muscle hypertrophy (Davies et al, 2003; Holgateet al, 2000). It is unknown whether this remodeling is due to or occursin parallel with the inflammatory response.

Little is known about the biology of IgA in asthma. However, upperairway infections are well known to frequently exacerbate the airflowobstruction that occurs in patients with asthma and other lungdiseases—a situation where the concentration of immunoglobulins in theairways increases. If ASM indeed possess receptors for IgA that alterASM biology or function, then a new pathophysiological explanationarises for these infectious exacerbations of asthma. In addition, thepresence or absence of such a receptor on ASM might account, at least inpart, for the variety of clinical manifestations and therapeuticresponsiveness amongst patients with asthma. Furthermore, a betterunderstanding of the cell and molecular biology of such receptors andtheir biology in ASM could lead to a novel approach for the diagnosisand treatment of asthma.

Immunoglobulin A (IgA) abundantly coats the enormous surface area of themucosal epithelium, which measures about 300-400 m² in adult humans.Total IgA transport is roughly 5-15 gms per day in an adult human with15% going into airway secretions (Childers et al, 1989). In fact, IgAcomprises 5-10% of the total protein in bronchoalveolar lavage fluid(Bell et al 1981). Despite its abundance, relatively little is knownabout the mechanism of action of IgA in host immune defense and immunetolerance. The mucosal epithelium is physically vulnerable to continuousexposure to potentially infectious agents, such as bacteria, viruses,fungi and parasites, as well as to substances in the environment ordiet. IgA is one of the most important proteins protecting the mucosalepithelium that guards the internal environment from the outside world.Elevated concentrations of IgA have been identified in induced sputumfrom asthmatics in contrast to that from healthy people (Louis et al,1997; Nahm and Park, 1997; Nahm et al, 1998). Furthermore, increasedlevels of specific IgA antibodies to both allergen and bacterial antigenhave been measured in induced sputum from asthmatics (Nahm et al, 1998).

Infections of the upper airway stimulate submucosal B cells to increaseproduction of specific pIgA. Furthermore, if infectious pathogensresulted in the breakdown of the epithelial barrier, then cells in thesubepithelial layers, such as those of ASM, become exposed to increasedconcentrations of pIgA as well as sIgA. Although airway inflammation andbronchoconstriction involve multifactorial and complex processes, if ASMpossess receptors for IgA that are activated by different isoforms ofIgA, then a novel pathophysiological mechanism is proposed to accountfor (1) the deterioration in airflow obstruction during infectiousexacerbations in patients with asthma, (2) the induction of asthma inpredisposed individuals, and (3) the temporary development of bronchialhyperreactivity in non-asthmatics (i.e. reactive airways dysfunctionsyndrome).

Polymeric Immunoglobulin Receptor

IgA exists in different isoforms (Mestecky et al, 1999). B lymphocytesresiding in submucosal tissues produce similar proportions of polymericIgA1 and IgA2 subclasses, secreting at least two IgA molecules linkedtogether by a J chain. Epithelial cells of the respiratory andgastrointestinal tracts abundantly express the polymeric immunoglobulinreceptor (pIgR) which transfers polymeric IgA from the submucosa (thebasolateral surface of the epithelium) to the lumenal (apical) surface(Mostov et al, 1995; Mostov and Kaetzel, 1999). At the apical surface,proteolytic cleavage of the pIgR releases secretory component (SC) boundto pIgA into mucosal secretions, called secretory IgA (sIgA). SCstabilizes sIgA from proteolytic degradation by bacterial enzymes andhelps neutralize pathogens, especially viruses. sIgA in mucosalsecretions is the first line of defense, acting to bind microorganismsand thereby limiting adhesion and colonization. IgA may neutralizeviruses and bacterial toxins by binding to antigenic determinantsimportant in the microorganism's interaction with cellular receptors.Additional roles for sIgA are postulated to include transport of immunecomplexes out through the epithelial surface by the pIgR. IgA istherefore a very important first line of host immune defense at mucosalsurfaces.

In contrast to mucosal secretions where sIgA prevails, the predominantform of IgA in human serum is monomeric IgA (mIgA) from B lymphocytes inthe bone marrow and spleen. While the pIgR will selectively mediatetransport of polymeric IgA across epithelial cells, this receptor doesnot bind monomeric IgA. IgA present in secretions therefore differs inbiochemical properties from IgA found in serum. The polymerization stateand the presence of SC might be expected to result in unique effectorfunctions for different forms of IgA depending on the site of productionand intended point of action.

Binding of pIgA to the pIgR on mucosal epithelial cells occurs via theJ-chain, which covalently links the IgA molecules together as dimers andmultimers (Johansen et al, 2001). In addition, pIgA, which is heavilyglycosylated, can bind to asialoglycoprotein receptors on liver cells.In contrast, binding to white blood cells (neutrophils, eosinophils,monocytes/macrophages) occurs by attachment of the Fc portion of IgA toFcαR (also known as CD89) expressed on these cells.

Human pIgR can also transport polymeric IgM, which contains J-chain andincreases in concentration during times of acute infections.

Fc-Alpha Receptors for IgA

Binding to white blood cells (neutrophils, eosinophils,monocytes/macrophages) occurs by attachment of the Fc portion of IgA toFc-alpha receptors (FcαR; also known as CD89) expressed on these cells(Kerr and Woof, 1999; Morton et al, 1996). Neutrophils andmonocytes/macrophages constitutively express FcαR as a 55-75 kd protein,while eosinophils express FcαR as a 70-100 kd protein with increasedglycosylation (Albrechtsen et al, 1988; Monteiro et al, 1990). FcαRexpression on monocytes and neutrophils increases in response to TNF-α,IL-1, GM-CSF, LPS or phorbol esters; IFN-γ and TGF-β1 decreaseexpression (discussed in Deo et al, 1998). The gene for FcαR is locatedon chromosome 19 and encodes several alternatively spliced isoforms ofthe receptor's α-chain (55-110 kD; Morton et al, 1996). FcαR can triggerrelease of inflammatory mediators and phagocytosis of IgA-coatedparticles (Yeaman and Kerr, 1987; Patry et el, 1995). IgA-coatedneutrophils and macrophages phagocytose particles, bacteria and immunecomplexes more efficiently than uncoated cells. Although theconcentration of the predominantly monomeric IgA in blood is high enoughto completely saturate the thousands of FcαR on neutrophils, mIgA willnot trigger signal transduction in PMNs unless the receptors arecrosslinked (Stewart et al., 1994). The pIgA and sIgA have the potentialto crosslink FcαR on cell surfaces due to their polymeric composition.So, during times of infection when submucosal B cells are stimulated toincrease production of specific pIgA, myeloid cells recruited to sitesof inflammation are better prepared for their functions in the mucosallumen. FcαR-induced calcium release and subsequent cytokine productiondepend on association with the FcR γ-chain (Morton et al, 1995). In vivostudies in transgenic mice show that while FcR γ chain is important forFcαR-triggered phagocytosis, CR3 (CD11b/CD18) is required forFcαR-mediated antibody-dependent cellular cytotoxicity (van Egmond etal, 1999).

FcαR may play a role in cancer in addition to its function againstmicrobial pathogens: IgA antitumor antibodies or bispecific antibodiesdirected to FcαR and tumor antigens effectively lyse tumor cells (Deo etal, 1998). Deo's work and that of others highlight FcαR as a potentialimmunotherapeutic target of malignant and infectious diseases (Valeriuset al, 1997). The novel finding of the FcαR on mesenchymal cells,including synovial fibroblasts and airway smooth muscle cells, thusindicates that targeting this receptor would be a promising and noveltherapeutic approach for inflammatory diseases, such as arthritis andasthma.

SUMMARY OF THE INVENTION

The inventors have unexpectedly found that the polymeric immunoglobulinreceptor (pIgR) and the Fc alpha receptor are expressed in human airwaysmooth muscle (ASM) cell cultures. The inventors have also shown thatincubation of ASM with the ligand for pIgR (pIgA) causes a rise inintracellular calcium concentrations that is unique in that the responseis delayed, sustained, oscillates and increases the sensitivity of ASMto subsequent stimulation with histamine. Incubation with mIgA (whichdoes not bind pIgR) does not cause this effect on intracellular calciumconcentrations in ASM. Smooth muscle responses to IgA have never beendescribed before.

The inventors have also found that the Fc alpha receptor (FcαR) isexpressed on synovial fibroblasts from patients with rheumatoidarthritis (RA) and osteoarthritis (OA). Furthermore, the inventors haveshown that incubating synovial fibroblasts with IgA causes an increasein NFκB DNA binding, and enhances TNF-alpha-induced ICAM-1 proteinexpression, and TNFα-induced IL-8 and RANTES mRNA expression. As aresult, inhibiting signalling through IgA receptors may be an effectivemeans of treating arthritis and other inflammatory diseases.

Fibroblasts, smooth muscle cells and endothelial cells are mesenchymalcells. The inventors are the first to show the presence of IgA receptorson these mesenchymal cells. Modulating these receptors are useful foraltering physiological responses in mesenchymal cells.

Accordingly, the present invention provides a method of modulating theinflammatory responses of a mesenchymal cell comprising administering toa cell or animal in need thereof an effective amount of an agent thatcan modulate an IgA receptor on a mesenchymal cell.

In a further embodiment, the present invention provides a method oftreating an inflammatory condition caused by IgA binding to an IgAreceptor on a mesenchymal cell comprising administering an effectiveamount of an IgA receptor antagonist to a patient in need thereof. Inone embodiment, the present invention provides a method of treating apatient with arthritis comprising administering an effective amount ofan IgA receptor antagonist to a patient in need thereof. In anotherembodiment, the present invention provides a method of treating apatient with asthma comprising administering an effective amount of anIgA receptor antagonist to a patient in need thereof.

The binding of IgA to an IgA receptor is known to induce cytosoliccalcium signalling and cause a number of calcium dependent effects.Accordingly, the present invention also provides a method of modulatingintracellular calcium signalling in a mesenchymal cell comprisingadministering to a cell or animal in need thereof an effective amount ofan agent that can modulate an IgA receptor on a mesenchymal cell. In oneembodiment, the present invention provides a method of inhibitingcytosolic calcium signalling in a mesenchymal cell comprisingadministering an effective amount of an IgA receptor antagonist to acell or animal in need thereof.

The discovery of IgA receptors on mesenchymal cells allows thedevelopment of methods to target delivery of a compound or substance toa mesenchymal cell. Accordingly, the present invention also includes amethod of delivering a substance to a mesenchymal cell comprisingadministering an effective amount of a conjugate comprising thesubstance coupled to an IgA receptor ligand to an animal or cell in needthereof.

The discovery of IgA receptors on synovial fibroblasts allowsdevelopment of diagnostic assays to detect IgA receptor-mediateddiseases or inflammatory conditions including arthritis (includingrheumatoid arthritis, osteoarthritis, spondyloarthropathies) and asthma,as well as other inflammatory diseases such as Crohn's disease,Ulcerative colitis, Behcet's disease, Sjogren's disease andvasculitides.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 shows immunofluorescence staining for FcαR in primary cellcultures of both RA and OA synovial fibroblasts FIG. 2 shows RT-PCRproduct bands for IgA-binding domain of FcαR in both RA and OA synovialfibroblasts.

FIG. 3 shows immunohistochemical staining of FcαR in synovial cells andendothelial cells in human RA and OA synovial tissue samples.

FIG. 4 shows a dose-dependent increase in NFκB activity in both RA andOA synovial fibroblasts treated with increasing concentrations of pIgA.

FIG. 5A is a graph showing that IgA increases TNFα-induced ICAM-1protein expression on RA synovial fibroblasts by cell ELISA.

FIG. 5B is a graph showing that an immune complex consisting of pIgA andan antibody to the alpha chain of IgA increases baseline RA synovialfibroblast expression of ICAM-1, in contrast to either reagent alone.

FIG. 6 is a graph showing that IgA further increases TNF-alpha inducedgene expression in rheumatoid arthritis synovial fibroblasts by RNaseprotection assay.

FIG. 7 is a graph showing that IgA further increases TNF-alpha inducedgene expression in osteoarthritis synovial fibroblasts by RNaseprotection assay.

FIG. 8A shows immunofluorescence staining for pIgR protein inserum-starved ASM using a rabbit antibody to human secretory component.

FIG. 8B shows immunofluorescence staining for the alpha chain of IgA inserum-starved ASM pre-incubated overnight with pIgA (live-cell uptake)in contrast to pre-incubation with mIgA.

FIG. 8C shows live uptake staining for the rabbit IgG antibody to humansecretory component in contrast to pre-incubation of live ASM cells withan irrelevant rabbit IgG.

FIG. 9 shows immunofluorescence staining for FcαR in non-starving ASMcells (upper panel) and that this staining is enhanced by pre-incubatinglive cells with either pIgA (middle panel) or mIgA (lower panel).

FIG. 10 shows a western blot for pIgR protein in non-starving human ASMthat is still present after 6 days and 12 days of serum starvation(upper panel). This blot was stripped and re-probed with aphosphotyrosine antibody to show that the pIgR protein in ASM is lessphosphorylated with serum deprivation.

FIGS. 11A show a pIgR band in serum-starved cells by RT-PCR using mRNAfrom ASM obtained from one human subject. MDCK cells transfected withhuman pIgR were used as a positive control. This figure also shows FcαRmRNA in serum-fed ASM and U937 cells used as a positive control cellline, in contrast to serum-starved ASM.

FIG. 11B shows the presence of pIgR mRNA in serum-starved and serum-fedASM obtained from a second subject by RT-PCR. FcαR mRNA is present inserum-fed ASM as determined by RT-PCR.

FIG. 12A shows calcium imaging studies in Fura-2 loaded serum-starvedASM stimulated with 12 μg/ml pIgA. At 80 min, pIgA causes an initialrise in cytosolic calcium concentrations and this effect becomessustained at 110 min. At this point, the calcium concentrationsoscillate. The oscillations abate and concentrations decrease 10 minafter washing off the pIgA.

FIG. 12B shows calcium imaging studies in Fura-2 loaded serum-starvedASM in buffer alone (time course study).

FIG. 13A shows calcium imaging studies in Fura-2 loaded serum-starvedASM pre-stimulated with histamine which is washed off prior to addinglow dose pIgA (0.12 μg/ml). Cytosolic calcium concentrations begin torise after 1 h and increase 4-6 fold with histamine stimulation afterwashing off pIgA.

FIG. 13B shows calcium imaging studies in Fura-2 loaded serum-starvedASM incubated with buffer alone (time control) after pre-stimulationwith histamine. These cells were re-exposed to histamine after 2 hoursin buffer and showed less than a 2-fold increase in cytosolic calciumconcentrations.

FIG. 13C shows calcium imaging studies in Fura-2 loaded serum-starvedASM stimulated with mIgA following pre-stimulation with histamine.(These serum-starved ASM do not express FcαR making mIgA a negativecontrol.) After washing off mIgA, the responses to histamine areaugmented by a factor of less than 2, as in FIG. 13B with buffer alone.

FIG. 13D shows calcium imaging studies in Fura-2 loaded serum- starvedASM stimulated with high dose pIgA 12 μg/ml following stimulation withhistamine as well as carbachol. Cytosolic calcium concentrations beginto rise 50 min later and go off-scale at 66 min with over a 30-foldincrease.

FIG. 14 shows a series of images obtained during Fura-2 calcium imagingstudies with serum-starved ASM stimulated with 12 μg/ml pIgA. The cellthat is circled in Frame 1 contracts and disappears by Frame 11.

FIG. 15 shows a western blot confirming that the different scFv clonesrecognize purified J-chain protein.

FIG. 16A is a graph showing that pIgA causes a dose-dependent rise incytosolic calcium in Fura2-loaded serum-starved human ASM grown in96-well fluorescence plates.

FIG. 16B shows that a mouse monoclonal anti-J-chain antibody decreasespIgA-induced increases in cystolic calcium concentration in Fura2-loadedserum-starved human ASM grown in 96-well fluorescence plates.

FIG. 17 is a graph showing that pIgA increases tension in isolatedstrips of dog tracheal smooth muscle as recorded at 5.5 h.

FIG. 18 is a graph showing increased tension responses to histamineafter incubating dog tracheal smooth muscle strips with pIgA for 4 h.

DETAILED DESCRIPTION OF THE INVENTION

I. Therapeutic Methods

As mentioned above, the present inventors have determined that IgAreceptors, such as Fc-alpha R, are present on RA and OA synovialfibroblasts as well as synovial tissue from arthritis patients and thatbinding the receptor stimulates inflammatory mediator production insynovial fibroblast cells. Non-diseased human skin fibroblasts andpulmonary fibroblasts do not express IgA receptors.

The present inventors have also determined that IgA receptors, includingpIgR and Fc-alpha R, are present on ASM cells and that binding thereceptor causes calcium signalling. Therefore the present inventionincludes all diagnostic and therapeutic methods for treating conditionsthat are mediated through modulation of signalling through IgA receptorson mesenchymal cells.

Broadly stated, the present invention provides a method of modulatingthe inflammatory responses of a mesenchymal cell comprisingadministering to a cell or animal in need thereof an effective amount ofan agent that can modulate an IgA receptor on a mesenchymal cell.

The present invention also includes a use of an effective amount of anagent that can modulate an IgA receptor on a mesenchymal cell tomodulate the inflammatory responses of a mesenchymal cell. The inventionfurther includes a use of an effective amount of an agent that canmodulate an IgA receptor on a mesenchymal cell for the manufacture of amedicament to modulate the inflammatory responses of a mesenchymal cell.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity as well as the induction orenhancement of a function or activity and interference with theinteraction between any isoform of IgA and its receptor such as pIgR orFcαR. For example, an agent that can modulate IgA receptors includesagents that can inhibit or block the signalling through this receptor(receptor antagonists) as well as agents that can induce or stimulatesignalling through the receptor (receptor agonists).

The term “IgA receptor” means any receptor on a mesenchymal cell thatcan bind an isoform of IgA. The receptor may also bind otherimmunoglobulins. In a preferred embodiment, the IgA receptor on themesenchymal cell is pIgR or FcαR.

The term “pIgR” as used herein denotes a polymeric immunoglobulinreceptor and means a receptor on cells that binds polymeric IgA (pIgA),dimeric IgA (dIgA) and polymeric IgM (pIgM) but not monomeric forms ofIgA. The term includes the pIgR that has been previously described onepithelial cells (Piskurich et al., J. Immuno. 154:1735-1747, 1995) aswell as any analogs, homologues, derivatives or variants (includingsplice variants) of the known pIgR molecules.

The term “FcαR” as used herein denotes the Fc-alpha receptor, also knownas CD89, and means a receptor on cells that binds any isoform of IgA byits Fc portion. The term includes the FcαR that has been previouslydescribed on white blood cells (Morton et al, Crit. Rev. Immunol. 16:423-440, 1996) as well as any analogs, homologues, derivatives orvariants (including splice variants) of the known FcαR molecules.

The term “mesenchymal cell” as used herein includes fibroblasts,synovial cells, smooth muscle cells and endothelial cells. Themesenchymal cell will express pIgR or a pIgR-like protein and/or theFc-alpha receptor. The mesenchymal cell is preferably a synovialfibroblast cell or an airway smooth muscle cell.

The term “a cell” as used herein includes a single cell as well as aplurality or population of cells. Administering an agent to a cellincludes both in vitro and in vivo administrations.

The term “animal” as used herein includes all members of the animalkingdom, including humans. Preferably, the animal to be treated is ahuman.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desired result,e.g. to modulate cell inflammatory responses or to modulate calciumsignalling.

The IgA receptor antagonist (such as a pIgR or FcαR antagonist) can beany agent that inhibits signalling through the IgA receptor and resultsin an inhibition of function caused by signalling through the receptorincluding an inhibition of cellular inflammation or an inhibition ofpIgR- or FcαR-mediated endocytosis. In one embodiment, the IgA receptorantagonist will inhibit the binding of pIgA to pIgR or FcαR onmesenchymal cells. The IgA receptor antagonist may be an antibody thatbinds, but does not activate the pIgR or FcαR on mesenchymal cells, andresults in an inhibition of the binding of IgA with the resultantinhibition of cellular inflammation or cytosolic calcium signalling.Other IgA receptor antagonists include anti-J chain antibodies thatmight interfere with the ability of pIgR to bind pIgA or pIgM orantibodies or ligands to the portion of the Fc-alpha part of IgA thatbinds to Fc-alpha receptor on airway smooth muscle cells. Examples ofother pIgR or FcαR antagonists are provided in Section II.

In one embodiment, the present invention provides a method of preventingor inhibiting the inflammatory responses of a mesenchymal cellcomprising administering an effective amount of an IgA receptorantagonist to a cell or animal in need thereof. The present inventionalso provides a use of an effective amount of an IgA receptor antagonistto prevent or inhibit the inflammatory responses of the mesenchymalcell. The invention further includes a use of an effective amount of anIgA receptor antagonist in the manufacture of a medicament to prevent orinhibit the inflammatory responses of a mesenchymal cell.

The term “preventing or inhibiting the inflammatory responses of amesenchymal cell” means that the inflammatory responses of themesenchymal cell in the presence of the IgA receptor antagonist isdecreased as compared to the level of inflammatory response in theabsence of the antagonist. Inflammatory responses of mesenchymal cellscan be measured using a variety of techniques known in the art includingthe techniques as described in the examples.

The methods of the invention can be used to treat any condition whereinit is desirable to modulate IgA receptor activity on mesenchymal cells.Such conditions include, but are not limited to, inflammatory diseasesincluding arthritides (including rheumatoid arthritis, osteoarthritis,spondyloarthropathies), asthma, Crohn's disease, ulcerative colitis,Behcet's disease, Sjogren's disease and vasculitides.

Accordingly, the present invention provides a method of treating aninflammatory condition caused by IgA binding to an IgA receptor on amesenchymal cell comprising administering an effective amount of an IgAreceptor antagonist to a patient in need thereof. The present alsoincludes the use of an effective amount of an IgA receptor antagonist totreat an inflammatory condition. The invention further includes a use ofan effective amount of an IgA receptor antagonist in the manufacture ofa medicament to treat an inflammatory condition.

As used herein, and as well understood in the art, “treating” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results can include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminishment of extent of disease, stabilized (i.e. notworsening) state of disease, preventing spread of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treating” can also mean prolonging survivalas compared to expected survival if not receiving treatment.

The inventors have shown that pIgA stimulates NF-κB activity in RAsynovial fibroblasts. As RA is characterized by increased NF-κB activityinhibiting the activity of this pro-inflammatory transcription factor(by inhibiting an IgA receptor) may be useful in treating arthritis. Theinventors have also shown that IgA increases TNF-α induced ICAM-1expression on synovial fibroblast and also increases TNF-α induced geneexpression in rheumatoid arthritis and osteoarthritis synovialfibroblasts. The inventors have also shown that immune complexes of IgAand IgG increase ICAM-1 expression in synovial fibroblasts. Accordingly,the present invention provides a method of treating a patient witharthritis comprising administering an effective amount of an IgAreceptor antagonist to a patient in need thereof. The invention furtherprovides the use of an effective amount of an IgA receptor antagonist totreat a patient with arthritis. The present invention further includes ause of an effective amount of an IgA receptor antagonist in themanufacture of a medicament to treat a patient with arthritis.

As mentioned previously, the binding of IgA to an IgA receptor inducesintracellular calcium signalling which further induces a variety ofcalcium dependent effects. Accordingly, the present invention provides amethod of preventing or inhibiting intracellular calcium signalling in amesenchymal cell comprising administering an effective amount of an IgAreceptor antagonist to a cell or animal in need thereof. The inventionfurther includes a use of an effective amount of an IgA receptorantagonist to prevent or inhibit intracellular calcium signalling in amesenchymal cell. The invention further includes a use of an effectiveamount of an IgA receptor antagonist in the manufacture of a medicamentto prevent or inhibit intracellular calcium signalling in a mesenchymalcell.

The term “preventing or inhibiting intracellular calcium signalling”means that the intracellular level of calcium in a mesenchymal cell inthe presence of the a IgA receptor antagonist is decreased as comparedto the level of intracellular calcium in cells in the absence of theagent. Calcium levels can be measured using a variety of knowntechniques including using fluorescence spectrophotometric and imagingtechniques.

Intracellular calcium signalling is important for several processes incell biology, including cell division, cytokine/chemokine/growth factorproduction, cell movement and contraction. Therefore, inhibiting calciumsignalling can inhibit a variety of calcium dependent effects.Accordingly, the present invention provides a method of inhibiting thecontraction of a mesenchymal cell comprising administering an effectiveamount of an IgA receptor antagonist to a cell or animal in needthereof.

The methods of the invention can be used to treat any condition whereinit is desirable to modulate IgA receptor (such as pIgR or FcαR) activityin order to prevent calcium signalling and thereby inhibit a variety ofcalcium dependent effects in airway smooth muscle cells. Such conditionsinclude, but are not limited to asthma, chronic bronchitis, acutebronchitis, bronchial hyperreactivity, chronic obstructive pulmonarydisease, emphysema, interstitial lung disease, bronchiectasis or airwayremodelling.

Accordingly, the present invention provides a method of treating acondition wherein it is desirable to inhibit a calcium dependent effectin an airway smooth muscle cell comprising administering an effectiveamount of an IgA receptor antagonist to an animal in need thereof. Theinvention also includes a use of an effective amount of an IgA receptorantagonist to treat a condition wherein it is desirable to inhibit acalcium dependent effect in an airway smooth muscle cell. The presentinvention also includes a use of an effective amount of an IgA receptorantagonist for the manufacture of a medicament to treat a conditionwherein it is desirable to inhibit a calcium dependent effect in anairway smooth muscle cell.

In a preferred embodiment, the method is useful in treating asthma. Itis possible that IgA-induced calcium signalling may directly influencesmooth muscle contraction and thereby contribute to bronchialhyperreactivity in asthma. It is also possible that IgA causesactivation of transcription factors (e.g. NFκB) important forinflammatory reactions and subsequent production of cytokines,chemokines, adhesion molecules and growth factors. As a result,inhibition of IgA-induced calcium signalling may greatly improve thedegree of inflammation in asthma as well as the airway remodellingdescribed in chronic asthmatics. Furthermore, it is possible that thisIgA-related phenomenon may account, at least in part, for the widevariety of clinical presentations and therapeutic responsiveness inpatients with asthma.

Accordingly, the present invention provides a method of treating apatient with asthma comprising administering an effective amount of anIgA receptor antagonist to a patient in need thereof. The inventionfurther provides the use of an effective amount of an IgA receptorantagonist to treat a patient with asthma. The present invention furtherincludes a use of an effective amount of an IgA receptor antagonist inthe manufacture of a medicament to treat a patient with asthma.

The present invention further provides a method of inhibiting theproduction of inflammatory mediators or growth factors comprising.administering an effective amount of an IgA receptor antagonist to acell or animal in need thereof. In a preferred embodiment, the methodinhibits the production of NF-κB. The present invention provides a useof an effective amount of an IgA receptor antagonist to inhibit theproduction of inflammatory mediators or growth factors. The presentinvention further provides a use of an effective amount of an IgAreceptor antagonist in the manufacture of a medicament to inhibit theproduction of inflammatory mediators or growth factors.

II. Agents that Modulate pIgR or FcαR

The finding by the present inventors that pIgR or FcαR are onmesenchymal cells allows the discovery and development of agents thatmodulate pIgR or FcαR for use in modulating diseases mediated through anIgA receptor, such as pIgR or FcαR, on mesenchymal cells.

The present invention includes the use of any and all agents thatmodulate pIgR or FcαR in the methods of the invention. The agent can beany type of substance, including, but not limited to, nucleic acids(including antisense oligonucleotides, proteins (including antibodies),peptides, peptide mimetics, carbohydrates, organic compounds, inorganiccompounds, small molecules, drugs, pIgR or FcαR ligands, soluble formsof pIgR or FcαR, pIgR or FcαR agonists, pIgR or FcαR antagonists, agentsthat inhibit pIgR or FcαR agonists, polymeric IgA (pIgA), dimeric IgA(dIgA) and polymeric IgM (pIgM) and fragments of these IgA or IgMmolecules. Examples of some of the agents that modulate pIgR or FcαR areprovided below.

(I) Antibodies

In one embodiment, the agent that can modulate pIgR is an antibody thatbinds to pIgR. Within the context of the present invention, antibodiesare understood to include monoclonal antibodies, polyclonal antibodies,antibody fragments (e.g., Fab, Fab′, F(ab′)₂, scFv and Fv fragments) andrecombinantly produced binding partners. Antibodies to pIgR may act aspIgR agonists or pIgR antagonists. For example, whole antibodies may actas pIgR agonists by stimulating the receptor while antibody fragmentsmay act as pIgR antagonists by blocking the ability of pIgR ligands(such as pIgA) to bind pIgR.

In one embodiment, the antibody is an antibody fragment that acts as apIgR antagonist. In a specific embodiment, the antibody fragment is asingle chain Fv antibody. Single chain-Fv fragments can be isolated froma phage display library. The preparation of scFv antibodies to pIgR isdescribed in Example 2. In Example 4, the inventors demonstrate that amouse monoclonal IgG1 anti-J chain antibody decreases the pIgA inducedincrease in cystolic calcium in airway smooth muscle cells.

In one embodiment, the agent that can modulate FcαR is an antibody thatbinds to FcαR. Within the context of the present invention, antibodiesare understood to include monoclonal antibodies, polyclonal antibodies,antibody fragments (e.g., Fab, Fab′, F(ab′)₂, scFv and Fv fragments) andrecombinantly produced binding partners. Antibodies to FcαR may act asFcαR agonists or FcαR antagonists. For example, whole antibodies may actas FcαR agonists by stimulating the receptor while antibody fragmentsmay act as FcαR antagonists by blocking the ability of FcαR ligands(such as mIgA or pIgA) to bind FcαR. Whereas several IgG, mousemonoclonal antibodies (A3, A59, A62 and A77) recognize the FcαR, onlythe mouse IgM monoclonal antibody My43 blocks the IgA binding site onthe receptor (Kerr and Woof, 1999). My43 which cross-links the FcαR hasalso been shown to trigger a respiratory burst in neutrophils andmonocytes as well as the release of calcium from intracellular stores inneutrophils (MacKenzie and Kerr, 1995; Shen, 1992; Stewart et al, 1994).

In one embodiment, the antibody is an antibody fragment that acts as aFcαR antagonist. In a specific embodiment, the antibody fragment is asingle chain Fv antibody. Single chain-Fv fragments can be isolated froma phage display library. The preparation of scFv antibodies to FcαR isdescribed in Example 2.

In another embodiment, the antibody is a pIgR agonist. Examples ofantibodies that are pIgR agonists include pIgA and pIgM. Antibodies tomesenchymal pIgR may be prepared using techniques known in the art suchas those described by Kohler and Milstein, Nature 256, 495 (1975) and inU.S. Pat. Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993, which areincorporated herein by reference. (See also Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988, which are also incorporated herein by reference).

In another embodiment, the antibody is a FcαR agonist. Examples ofantibodies that are FcαR agonists include mIgA and pIgA. Antibodies tomesenchymal FcαR may be prepared using techniques known in the art suchas those described by Kohler and Milstein, Nature 256, 495 (1975) and inU.S. Pat. Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993, which areincorporated herein by reference. (See also Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988, which are also incorporated herein by reference).

(ii) Antisense Oligonucleotides

In another embodiment, the agent that can modulate pIgR or FcαR is anantisense oligonucleotide that acts as a pIgR or FcαR antagonist,respectively, by inhibiting the expression of the pIgR or FcαR gene. Theterm “antisense oligonucleotide” as used herein means a nucleotidesequence that is complimentary to its target, e.g. the pIgR or FcαRgene. The sequence of the pIgR and FcαR genes are known in the art formany species, for example, see Piskurich et al., J. Immunol.154:1735-1747, 1995, and Maliszewski et al, J. Exp. Med. 172:1665-1672,1990.

The term “oligonucleotide” as used herein refers to an oligomer orpolymer of nucleotide or nucleoside monomers consisting of naturallyoccurring bases, sugars, and intersugar (backbone) linkages. The termalso includes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligonucleotides may be preferred over naturallyoccurring forms because of properties such as enhanced cellular uptake,or increased stability in the presence of nucleases. The term alsoincludes chimeric oligonucleotides which contain two or more chemicallydistinct regions. For example, chimeric oligonucleotides may contain atleast one region of modified nucleotides that confer beneficialproperties (e.g. increased nuclease resistance, increased uptake intocells), or two or more oligonucleotides of the invention may be joinedto form a chimeric oligonucleotide.

The antisense oligonucleotides of the present invention may beribonucleic or deoxyribonucleic acids and may contain naturallyoccurring bases including adenine, guanine, cytosine, thymidine anduracil. The oligonucleotides may also contain modified bases such asxanthine, hypoxanthine. 2-aminoadenine, 6-methyl, 2-propyl and otheralkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-azacytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Other antisense oligonucleotides of the invention may contain modifiedphosphorous, oxygen heteroatoms in the phosphate backbone, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. For example, the antisenseoligonucleotides may contain phosphorothioates, phosphotriesters, methylphosphonates, and phosphorodithioates. In an embodiment of the inventionthere are phosphorothioate bonds links between the four to six3′-terminus bases. In another embodiment phosphorothioate bonds link allthe nucleotides.

The antisense oligonucleotides of the invention may also comprisenucleotide analogs that may be better suited as therapeutic orexperimental reagents. An example of an oligonucleotide analogue is apeptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in the DNA (or RNA), is replaced with a polyamide backbonewhich is similar to that found in peptides (P. E. Nielsen, et al Science1991, 254, 1497). PNA analogues have been shown to be resistant todegradation by enzymes and to have extended lives in vivo and in vitro.PNAs also bind stronger to a complimentary DNA sequence due to the lackof charge repulsion between the PNA strand and the DNA strand. Otheroligonucleotides may contain nucleotides containing polymer backbones,cyclic backbones, or acyclic backbones. For example, the nucleotides mayhave morpholino backbone structures (U.S. Pat. No. 5,034,506).Oligonucleotides may also contain groups such as reporter groups, agroup for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an antisense oligonucleotide. Antisense oligonucleotides may alsohave sugar mimetics.

The antisense nucleic acid molecules may be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

(iii) Peptide Mimetics

The present invention also includes peptide mimetics of the pIgR or FcαRproteins. Such peptides may include competitive inhibitors, enhancers,peptide mimetics, and the like. All of these peptides as well asmolecules substantially homologous, complementary or otherwisefunctionally or structurally equivalent to these peptides may be usedfor purposes of the present invention.

“Peptide mimetics” are structures which serve as substitutes forpeptides in interactions between molecules (See Morgan et al (1989),Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimeticsinclude synthetic structures which may or may not contain amino acidsand/or peptide bonds but retain the structural and functional featuresof a pIgR peptide, or enhancer or inhibitor of the pIgR peptide. Peptidemimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc.Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptidesof a designed length representing all possible sequences of amino acidscorresponding to a peptide of the invention.

Peptide mimetics may be designed based on information obtained bysystematic replacement of L-amino acids by D-amino acids, replacement ofside chains with groups having different electronic properties, and bysystematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptoidsmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

(iv) Other Substances

In addition to antibodies and antisense oligonucleotides, othersubstances that can modulate pIgR or FcαR can also be identified andused in the methods of the invention. In one embodiment, the pIgR orFcαR modulator is a protein or peptide that can bind to pIgR or FcαR.The pIgR- or FcαR-binding peptides may be isolated by assaying a samplefor peptides that bind to pIgR or FcαR. Any assay system or testingmethod that detects protein-protein interactions may be used includingco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns may be used. Biological samples andcommercially available libraries may be tested for pIgR- or FcαR-bindingpeptides. For example, labelled pIgR or FcαR may be used to probe phagedisplay libraries. In addition, antibodies that bind pIgR or FcαR may beused to isolate other peptides with pIgR or FcαR binding affinity. Forexample, labelled antibodies may be used to probe phage displaylibraries or biological samples. Additionally, a DNA sequence encoding apIgR protein may be used to probe biological samples or libraries fornucleic acids that encode pIgR- or FcαR-binding proteins.

Substances which can bind pIgR or FcαR may be identified by reactingpIgR or FcαR, respectively, with a substance which potentially binds topIgR or FcαR, then detecting if complexes between the respectivereceptor and the substance have formed. Substances that bind pIgR orFcαR in this assay can be further assessed to determine if they areuseful in modulating or inhibiting pIgR or FcαR and useful in thetherapeutic methods of the invention.

Accordingly, the present invention also includes a method of identifyingsubstances which can bind to mesenchymal pIgR or FcαR comprising thesteps of:

(a) reacting pIgR or FcαR on a mesenchymal cell and a test substance,under conditions which allow for formation of a complex between the pIgRor FcαR and the test substance, and

(b) assaying for complexes of pIgR or FcαR and the test substance, forfree substance or for non complexed pIgR or FcαR, wherein the presenceof complexes indicates that the test substance is capable of bindingpIgR or FcαR.

Conditions which permit the formation of substance and IgA receptorcomplexes may be selected having regard to factors such as the natureand amounts of the substance and the protein.

The substance-IgA receptor complex, free substance or non-complexedproteins may be isolated by conventional isolation techniques, forexample, salting out, chromatography, electrophoresis, gel filtration,fractionation, absorption, polyacrylamide gel electrophoresis,agglutination, or combinations thereof. To facilitate the assay of thecomponents, antibody against pIgR or FcαR or the substance, or labelledpIgR or FcαR, or a labelled substance may be utilized. The antibodies,pIgR or FcαR, or substances may be labelled with a detectable substance.

The pIgR or FcαR gene or protein may be used as a target for identifyinglead compounds for drug developments. The invention therefore includesan assay system for determining the effect of a test compound orcandidate drug on the activity of the pIgR or FcαR gene or protein.

Accordingly, the present invention provides a method for identifying acompound that modulates mesenchymal pIgR or FcαR activity comprising:

(a) incubating a test compound with a mesenchymal pIgR or FcαR proteinor a nucleic acid encoding a mesenchymal pIgR or FcαR protein; and

(b) determining the effect of the test compound on the pIgR or FcαRprotein activity or pIgR or FcαR gene expression and comparing with acontrol (i.e. in the absence of a test compound) wherein a change in thepIgR or FcαR protein activity or pIgF3 or FcαR gene expression ascompared to the control indicates that the test compound is a potentialmodulator of the pIgR or FcαR gene or protein.

In one embodiment, pIgR or FcαR activity may be assessed by measuringintracellular calcium levels as previously described.

III. Compositions

The present invention also includes pharmaceutical compositionscontaining the agents that can modulate or inhibit pIgR or FcαR for usein the methods of the invention. Accordingly, the present inventionprovides a pharmaceutical composition for modulating the inflammatoryresponses of a mesenchymal cell comprising an effective amount of anagent that can modulate an IgA receptor in admixture with a suitablediluent or carrier. The present invention also includes a pharmaceuticalcomposition for preventing or inhibiting the inflammatory responses of amesenchymal cell comprising an effective amount of an IgA receptorantagonist in admixture with a suitable diluent or carrier. The presentinvention further provides a pharmaceutical composition for preventingor treating arthritis comprising an effective amount of an IgA receptorantagonist in admixture with a suitable diluent or carrier. In apreferred embodiment, the IgA receptor antagonist is a pIgR or FcαRantagonist.

Such pharmaceutical compositions can be for intralesional, intravenous,topical, rectal, parenteral, local, inhalant or subcutaneous,intradermal, intramuscular, intra-articular, intrathecal,transperitoneal, oral, and intracerebral use. The composition can be inliquid, solid or semisolid form, for example pills, tablets, creams,gelatin capsules, capsules, suppositories, soft gelatin capsules, gels,membranes, tubelets, solutions or suspensions.

The pharmaceutical compositions of the invention can be intended foradministration to humans or animals. Dosages to be administered dependon individual needs, on the desired effect and on the chosen route ofadministration.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, the active compound or substance in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids. The pharmaceutical compositions may additionallycontain other agents such as other agents that can modulate or inhibitcell inflammatory responses or that are used in treating inflammatoryconditions such as arthritis and asthma.

IV. Targeted Delivery

The finding by the present invention that pIgR and FcαR are onmesenchymal cells allows the development of methods to target thedelivery of substances directly to mesenchymal cells. Accordingly, thepresent invention provides a method of delivering a substance to amesenchymal cell comprising administering an effective amount of aconjugate comprising the substance coupled to an IgA receptor ligand toan animal or cell in need thereof.

The substance can be any substance that one wishes to deliver, includingtherapeutics and diagnostics to a mesenchymal cell in a specificembodiment, the substance is useful in treating an inflammatorycondition such as arthritis.

The ligand can be any molecule that can bind the IgA receptor, includingpIgA or pIgM, as well as the ligands described in Section II.

The substance may be coupled to the IgA receptor ligand either directlyor indirectly. In direct coupling, the substance and ligand arephysically linked such as by covalent binding or physical forces such asvan der Waals or hydrophobic interactions. In indirect coupling, thesubstance and ligand are joined through another molecule or linker. Asone example, the substance and ligand may be joined through a bispecificantibody that binds both the substance and linker.

Conjugates of the substance arid the IgA receptor ligand may be preparedusing techniques known in the art. There are numerous approaches for theconjugation or chemical crosslinking of proteins and one skilled in theart can determine which method is appropriate for the substance to beconjugated. The method employed must be capable of joining the substancewith the IgA receptor ligand without interfering with the ability of theligand to bind to the IgA receptor and without significantly alteringthe activity of the substance. If the substance and ligand are bothproteins, there are several hundred crosslinkers available in order toconjugate the substance with the ligand. (See for example “Chemistry ofProtein Conjugation and Crosslinking”. 1991, Shans Wong, CRC Press, AnnArbor). The crosslinker is generally chosen based on the reactivefunctional groups available or inserted on the substance. In addition,if there are no reactive groups a photoactivatible crosslinker can beused. In certain instances, it may be desirable to include a spacerbetween the substance and the ligand. In one example, the ligand andsubstance may be conjugated by the introduction of a sulfhydryl group onthe ligand and the introduction of a cross-linker containing a reactivethiol group on to the substance through carboxyl groups (Wawizynczak, E.J. and Thorpe, P. E. in Immunoconjugates: Antibody Conjugates inRadioimaging and Therapy of Cancer, C. W. Vogel (Ed.) Oxford UniversityPress, 1987, pp. 28-55.; and Blair, A. H. and T. I. Ghose, J. Immunol.Methods 59:129, 1983).

In another embodiment, the protein ligand and substance may be preparedas a fusion protein. Fusion proteins may be prepared using techniquesknown in the art. In such a case, a DNA molecule encoding the IgAreceptor ligand is linked to a DNA molecule encoding the substance. Thechimeric DNA construct, along with suitable regulatory elements can becloned into an expression vector and expressed in a suitable host.

The conjugates of the invention may be tested for their ability to entermesenchymal cells and provide the desired pharmacological effect usingin vitro and in vivo models.

V. Diagnostic Assays

The finding by the present inventors that airway smooth muscle cellshave IgA receptors (such as pIgR and FcαR) allows the development ofdiagnostic assays to detect IgA mediated diseases. Such diagnosticassays can facilitate the development of tailored therapies for suchdiseases. In one example, patients can undergo bronchial challengetesting to determine the presence of IgA-mediated bronchialhyperreactivity. Methacholine challenge and histamine challenge testingare examples of currently used tests to evaluate respiratory symptoms ofcough, wheeze and shortness of breath, and to detect nonspecificbronchial hyperresponsiveness as demonstrated by exaggeratedbronchoconstriction to inhaled methacholine or histamine. Pre- andpost-challenge pulmonary function test measurements of forced expiratoryvolume at 1 second (FEV1) and forced vital capacity (FVC) could be doneevery 5-10 minutes following bronchial challenge with an agonist to IgAreceptors that would result in increased intracellular calciumconcentrations in ASM and consequent bronchoconstriction. The IgAreceptor agonist can be any substance that will increase airway smoothmuscle cytosolic calcium concentrations and contraction which wouldmanifest clinically as bronchoconstriction. This bronchoconstriction canbe detected subjectively by hearing wheezing on chest auscultation, orobjectively by measuring a reduced FEV1 by standard spirometry orreduced peak flow. For example, bronchoconstriction may be detected by adrop in FEV1. IgA-mediated bronchial hyperreactivity could be gradedaccording to the concentration of the test substance that results in a20% fall in baseline FEV1. Accordingly, the present invention provides amethod of detecting IgA mediated bronchial hyperreactivity comprising:

(a) administering an IgA receptor agonist to a patient; and

(b) detecting bronchoconstriction in the patient wherein an increase inbronchoconstriction as compared to a control indicates that the patienthas IgA-mediated bronchial hyperreactivity.

Another example of a diagnostic test would be to perform a nonspecificbronchial challenge test with either methacholine or histamine on oneday, and then repeat this challenge on another day after pretreatingpatients with an IgA receptor agonist. IgA-mediated bronchialhyperreactivity would be detected by a significant increase sensitivityto the non-specific bronchoconstrictor (i.e. a lower dose ofmethacholine or histamine induces the 20% fall in FEV1 from baselinemeasurement). Another example would be to perform a bronchial challengetest with both a nonspecific bronchoconstrictor and an IgA receptoragonist.

Accordingly, the present invention provides a method of detectingIgA-mediated bronchial hyperreactivity comprising:

(a) administering an IgA-receptor agonist to a patient and detectingbronchoconstriction; and

(b) administering an IgA receptor agonist followed by a non-specificbronchoconstricting agent to the patient at a lower dose than when thenonspecific agent is administered alone and detectingbronchoconstriction wherein bronchoconstriction in step (a) and/orbronchoconstriction induced at a lower dose of the nonspecific agentadministered without the IgA receptor agonist in step (b) would indicatethat the patient has IgA-mediated bronchial hyperreactivity.

In a bronchial challenge test, a patient inhales increasing amounts ofthe agent. The patient's FEV1 and FVC are measured by spirometry 30-90seconds after inhaling the agent. If there is no significant (=20%change from baseline), the next dose is administered 5 minutes later,but no sooner than 5 minutes. The challenge is stopped when the patientdevelops a 20% drop in baseline FEV1 (—which indicatesbronchoconstriction—or FVC) or a specific maximum dose has been given(=8 mg/ml in the case of methacholine). (Higher doses of methacholinewill cause bronchoconstriction in nearly everyone. People who react todoses less than 8 mg/ml are categorized as having severe, moderate, mildor borderline hyperreactivity to the agent depending on the dose of theagent that causes the 20% drop in FEV1. These challenge tests are usedto diagnose bronchial hyperreactivity and monitor therapy. Note thatnarrowing might occur in very distal airways without narrowing in moreproximal airways and that this very distal narrowing might not result inchanges in FEV1 but would be suggested by a decrease in FEF_(25-75%)(forced expiratory flow during 25 to 75% of the forced vital capacity).

The finding by the present inventors that synovial fibroblasts andendothelial cells in synovial tissue from arthritis patients as well asairway smooth muscle cells have IgA receptors (such as pIgR and FcαR)allows the development of diagnostic assays to detect diseases mediatedthrough IgA binding to an IgA receptor on a mesenchymal cell. Suchdiagnostic assays can facilitate the development of tailored therapiesfor such diseases. Samples from patients can be obtained and tested forthe presence of IgA receptors, such as pIgR or FcαR, on mesenchymalcells. The sample can be any sample that contains a mesenchymal cellincluding synovial fibroblasts and synovial tissue, connective tissue,endothelial cells and blood vessels, smooth muscle cells, or primarycell cultures of these cells derived from a tissue biopsy. Patientsexpressing an IgA receptor may be treated with IgA receptor antagonistsas described above.

Accordingly, the present invention provides a method of detecting acondition associated with the activation of an IgA receptor on amesenchymal cell comprising assaying a sample for (a) a nucleic acidmolecule encoding an IgA receptor or a fragment thereof or (b) an IgAreceptor or a fragment thereof. The IgA receptor is preferably pIgR orFcαR. In one embodiment, the condition associated with the activation ofan IgA receptor on a mesenchymal cell is an Inflammatory condition suchas arthritides (including rheumatoid arthritis, osteoarthritis,spondyloarthropathies), asthma, Crohn's disease, ulcerative colitis,Behcet's disease, Sjogren's disease and vasculitides.

(i) Detecting Nucleic Acid Molecules Encoding IgA Receptors

Nucleotide probes can be prepared and used in the detection ofnucleotide sequences encoding an IgA receptor or fragments thereof insamples, preferably pIgR or FcαR. The probes can be useful in detectingthe presence of a condition associated with the activation of an IgAreceptor on a mesenchymal cell or monitoring the progress of suchconditions include inflammatory conditions including the arthritides(including rheumatoid arthritis, osteoarthritis, spondyloarthropathies),Crohn's disease, ulcerative colitis, Behcet's disease and Sjogren'sdisease and vasculitides. Accordingly, the present invention provides amethod for detecting a nucleic acid molecule encoding an IgA receptorcomprising contacting the sample with a nucleotide probe capable ofhybridizing with the nucleic acid molecule to form a hybridizationproduct, under conditions which permit the formation of thehybridization product, and assaying for the hybridization product.

The nucleotide probe may be labelled with a detectable substance such asa radioactive label which provides for an adequate signal and hassufficient half-life such as 32P, 3H, 14C or the like. Other detectablesubstances which may be used include antigens that are recognized by aspecific labelled antibody, fluorescent compounds, enzymes, antibodiesspecific for a labelled antigen, and chemiluminescence. An appropriatelabel may be selected having regard to the rate of hybridization andbinding of the probe to the nucleic acid to be detected and the amountof nucleic acid available for hybridization. Labelled probes may behybridized to nucleic acids on solid supports such as nitrocellulosefilters or nylon membranes as generally described in Sambrook et al,1989, Molecular Cloning, A Laboratory Manual (2nd ed.).

Nucleic acid molecules encoding an IgA receptor can be selectivelyamplified in a sample using the polymerase chain reaction (PCR) methodsand cDNA or genomic DNA. A nucleic acid can be amplified from cDNA orgenomic DNA using oligonucleotide primers and standard PCR amplificationtechniques. The amplified nucleic acid can be cloned into an appropriatevector and characterized by DNA sequence analysis. cDNA may be preparedfrom mRNA, by isolating total cellular mRNA by a variety of techniques,for example, by using the guanidinium-thiocyanate extraction procedureof Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is thensynthesized from the mRNA using reverse transcriptase (for example,Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda,Md., or AMV reverse transcriptase available from Seikagaku America,Inc., St. Petersburg, Fla.).

(ii) Detecting IgA Receptors

The presence of IgA receptors may be detected in a sample using IgAreceptor ligands that bind to the IgA receptor. IgA receptor ligands aredescribed above and include antibodies or other substances that can bindan IgA receptor. Accordingly, the present invention provides a methodfor detecting an IgA receptor comprising contacting the sample with aligand that binds to an IgA receptor which is capable of being detectedafter it becomes bound to the IgA receptor in the sample.

Ligands to an IgA receptor, such as antibodies specifically reactivewith an IgA receptor, or derivatives thereof, such as enzyme conjugatesor labeled derivatives, may be used to detect an IgA receptor in variousbiological materials. For example they may be used in any knownimmunoassays which rely on the binding interaction between an IgAreceptor, and an antibody thereof. Examples of such assays areradioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence,immunoprecipitation, latex agglutination, hemagglutination andhistochemical tests. Thus, the antibodies may be used to detect andquantify an IgA receptor in a sample in order to determine its role inparticular cellular events or pathological states, and to diagnose andtreat such pathological states, such as arthritis.

Cytochemical techniques known in the art for localizing antigens usinglight and electron microscopy may be used to detect an IgA receptor.Generally, an antibody of the invention may be labelled with adetectable substance and an IgA receptor may be localised in tissuebased upon the presence of the detectable substance. Examples ofdetectable substances include various enzymes, fluorescent materials,luminescent materials and radioactive materials. Examples of suitableenzymes include horseradish peroxidase, biotin, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material includeradioactive iodine I-125, I-131 or 3-H. Antibodies may also be coupledto electron dense substances, such as ferritin or colloidal gold, whichare readily visualised by electron microscopy.

Indirect methods may also be employed in which the primaryantigen-antibody reaction is amplified by the introduction of a secondantibody, having specificity for the antibody reactive against an IgAreceptor. By way of example, if the antibody having specificity againstan IgA receptor is a rabbit IgG antibody, the second antibody may begoat anti-rabbit gamma-globulin labelled with a detectable substance asdescribed herein.

Where a radioactive label is used as a detectable substance, an IgAreceptor may be localized by autoradiography. The results ofautoradiography may be quantitated by determining the density ofparticles in the autoradiographs by various optical methods, or bycounting the grains.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1

Primary Cell Cultures of RA and OA Synovial Fibroblasts Express FcαR

(a) FcαR protein expression was studied in primary cultures of RA and OAsynovial fibroblasts by immunofluorescence. Tissues were obtained frompatients undergoing joint replacement surgery and were digested withcollagenase (1 mg/ml) and hyaluronidase (0.05 mg/ml) (Sigma) in Hanks'balanced salt solution (Gibco) for 1-2 h at 37 C. Cells were washed with10% fetal bovine serum (FBS) in RPMI 1640 (Gibco), spun and grown inthis same media for overnight culture at 37 C. Subsequently, cells werecultured in DMEM with 10% FBS, penicillin and streptomycin. Both RA andOA cells showed staining for FcαR using either a mouse (Santa Cruz) orgoat (Santa Cruz) antibody to FcαR and the appropriate FITC-conjugatedsecondary antibody. FIG. 1 shows data obtained with the goat antibody toFcαR (Santa Cruz) (representative experiment of n=4 different subjectswith RA and n=3 for OA).

Primary Cell Cultures of RA and OA Synovial Fibroblasts Express FcαRmRNA by RT-PCR

(b) FcαR mRNA expression was confirmed in both RA (n=9 differentsubjects) and OA (n=4) synovial fibroblasts by RT-PCR. FIG. 2 showsrepresentative data from a total of 3 different patients with RA and 3with OA. Primers for the the IgA binding domain of FcαR (sense: 5′ CCTCAG TCT GGG GCT TTC TTU 3′; antisense: 5′ CTT GTT TGC GTC CAT GTG GTC3′) were used. The bands obtained from the RT-PCR product were DNAsequenced and confirmed to be sequences of their respective IgAreceptors. These results confirm that RA and OA synovial fibroblastsexpress mRNA for FcαR.

Synovial Tissue from Patients with Arthritis Express Receptors for IgA.

Acetone-fixed frozen sections of synovial tissue from arthritis patientsundergoing joint replacement surgery were stained for IgA receptorexpression. We have studied a total of 4 patients with RA and 3 otherpatients with OA using a goat polyclonal antibody to FcαR (Santa Cruz),and an HRP-conjugated secondary antibody (Jackson). FIG. 3 shows arepresentative slide confirming the presence of FcαR in synovial tissuefrom one patient each with RA or OA. Positive staining for FcαR can beseen in synovial cells within the tissue as well as on endothelial cellsof blood vessels These results confirm that IgA receptor expressionoccurs in vivo in arthritis tissue as well as primary cell cultures ofsynovial fibroblasts. These results also show that a third type ofmesenchymal cell—an endothelial cell—also expresses an IgA receptor,

IgA Stimulates NF-κB DNA Binding in RA and OA Synovial Fibroblasts.

RA is characterized by increased activity of the pro-inflammatorytranscription factor, NFκB, in synovial fibroblasts. Both RA and OA arechronic inflammatory conditions, but RA is an autoimmune inflammatorydisease. To determine whether expression of IgA receptors might play arole in the inflammation of RA and OA, we asked whether pIgA stimulatesNFκB DNA binding in RA and OA synovial fibroblasts. We found that pIgAinduced a dose-dependent increase in NFκB activity in both RA and OAsynovial fibroblasts by DNA electromobility gel shift assay (EMSA) (FIG.4). EMSA was performed using the Promega gel shift assay system. TheNFκB consensus oligonucleotide (5′]AGT TGA GGG GAC TTT CCC AGG C-3′)representing the p65 subunit was end-labeled with [γ-³²P]ATP using T4polynucleotide kinase. EMSA was done using 5 μg of nuclear extractproteins and labeled oligonucleotide. The protein-DNA comples wasseparated on polyacrylamide gel, which was then exposed toautoradiographic film. This effect of IgA on increasing NFkB DNA bindingin synovial fibroblasts has never been described and has majorimplications for the role of IgA receptors in RA and OA.

These results implicate synovial FcαR in the inflammatory processes ofRA and OA.

Example 2

scFv Selection Methods and Results:

A scFv phage library was reconstituted by pooling several first roundsof selection that the inventor had previously used. The scFv phagelibrary that was originally used is described in: Sheets M D,Amersdorfer P, Finnern R, Sargent P, Lindquist E, Schier R, Hemingsen G,Wong C, Gerhart J C, Marks J D, Lindqvist E., Efficient construction ofa large nonimmune phage antibody library: the production ofhigh-affinity human single-chain antibodies to protein antigens. (ProcNatl Acad Sci USA. May 26, 1998;95(11):6157-62.).

TG-1/pHen/phage^(1st round) scFv. These selections were to domain of ratpIgR; and to cell selections for pIgR with MDCK cells transfected withrabbit pIgR and attempted in 12 different ways. These first round TG-1from 13 different selections were combined and grown for isolatingphage. These phage were used as the “reconstituted” phage library ofscFv.

A. For Selections Against a Mesenchymal FcαR:

1. Coat 3 immunotubes with mIgA (Biolynx; 6.5 λ/3 ml PBS) and block with2% milk.

2. Preclear reconstituted phage library twice with 2 of the coatedimmunotubes.

3. Incubate SDS lysates from non-serum-starved mesenchymal cells (e.g.airway smooth muscle cells, ASM, purchased from Biowhittaker) with the3rd coated tube (—to bind the putative ASM FcαR to the mIgA).

4. Incubate precleared phage with the 3rd tube.

5. Wash extensively (15-20) with PBS and elute the bound phage with 1%TEA (triethanolamine). Neutralize the high pH with 1M Tris pH 7.4.

6. Infect TG-1 E. coli with the phage, and grow.

7. Expand and rescue phage to repeat procedure 2 more times.

8. After round 3, randomly pick 96 colonies and screen these for scFvproduction (dot blot) and cell ELISAs using (a) U937 cells(myelomonocytic cell line that highly expresses the FcαR); and (b) ASM,both cell lines grown in a 96-well plate.

Results:

8 positives by ASM ELISA (OD450>0.2); 3 positives by U937 cell ELISA

-   -   BstN1 DNA digest of pcr products from the 3 clones showed unique        patterns, suggesting isolation of 3 different scFv        B. For selections against a mesenchymal pIgR:

1. Coat 3 immunotubes with pIgA (10λ myeloma serum/3 ml PBS) and blockwith 2% milk PBS.

2. Preclear reconstituted phage library twice with 2 of the coatedimmunotubes.

3. Incubate SDS lysates from non-serum-starved mesenchymal cells (e.g.ASM, purchased from Biowhiftaker) with the 3rd coated tube (—to bind theputative ASM pIgR to the pIgA).

4. Incubate precleared phage with the 3rd tube.

5. Wash extensively (15-20) with PBS and elute the bound phage with 1%TEA. Neutralize with 1M Tris pH 7.4.

6. Infect TG-1 with the phage, and grow.

7. Expand and rescue phage to repeat procedure 2 more times.

8. After round 3, randomly pick 96 colonies and screen these for scFvproduction (dot blot) and cell ELISA using ASM and CALU-3 cells grown ina 96-well plate.

Results:

45 positives by ASM ELISA (includes 6 that were negative on CALU-3); 55positives by CALU-3 ELISA.

-   -   Also screened by ELISA with hurrian milk which contains        secretory component, the extracellular part of pIgR, and found        26 positives (used OD450>0.4 with background reading of ˜0.1);        screened by ELISA with fetal calf serum-coated plate, and found        46 positives; rabbit anti-human SC antibody (Dako) used as        positive control antibody    -   BstN1 DNA digest of pcr products from the all positive clones        showed 12 unique patterns, suggesting isolation of 12 different        scFv        C. For Selections Against J-Chain:

Dr. Jiri Mestecky sent his PET32 plasmid containing the J-chain proteinfused to thioredoxin and containing an IgA protease cleavage site and a6His tag for purification (Symersky et al, 2000). This plasmid wasinfected into BL21 E. coli which were induced to produce theJ-chain-thioredoxin fusion protein. This was purified by IMAC on anickel resin.

1. Coat 2 immunotubes with thioredoxin (Sigma; 10 μg/ml) and block with2% milk/PBS.

2. Coat 1 immunotube with the purified J-chain fusion protein, thenblock with 2% milk/PBS.

3. Preclear reconstituted phage library twice with the 2thioredoxin-coated immunotubes.

4. Incubate precleared phage with the 3rd tube coated with the J-chainfusion protein.

5. Wash extensively (15-20) with PBS and elute the bound phage with TEA(triethanolamine). Neutralize the high pH with Tris buffer.

6. Infect [TG-1] E. coli with the phage, and grow.

7. Expand and rescue phage to repeat procedure 2 more times.

8. After round 3, randomly pick 96 colonies and screen these for scFvproduction (dot blot) and protein ELISA using one 96-well plate coatedwith thioredoxin and one plate coated with the J-chain fusion protein.(—To ensure that the scFv selected bind to J-chain and not tothioredoxin.)

Results:

30 positives by J-chain ELISA; none bound the thioredoxin-coated plate.(Background OD450 was ˜0.07; chose OD450>0.2 to be positive.)

14 of these were induced to produce scFv (which contain a myc epitopetag) and all recognized J-chain by western blotting (mouse monoclonalanti-J-chain from InnoGenex was used as positive control); 9E10(anti-myc mouse monoclonal antibody and anti-mouse HRP alone used asnegative control). (FIG. 15)

-   -   BstN1 DNA digest of pcr products from all positive clones showed        5 unique patterns, suggesting isolation of 5 different scFv.    -   Of these 5 unique scFv binders to j-chain, 1 binds both protein        L and protein A and 3 bind protein A. This characteristic        constitutes additional evidence that these scFv differ from each        other and provides another means to purify these scFv for        further testing.

Example 3

Methods

Cell Culture:

Primary human airway smooth muscle (ASM) cells from three differentsubjects were purchased from Clonetics (San Diego, Calif., USA) andgrown in smooth muscle cell basal medium (SmBM) (Clonetics) supplementedwith the smooth muscle cell SingleQuots (Clonetics), which containing0.5 ng/ml human recombinant Epidermal Growth Factor (hEGF), 5 μg/mlinsulin, 1 μg/ml human recombinant Fibroblast Growth Factor (hFGr), 50μg/ml Gentamicin and 50 ng/ml Amphotericin-B, and 5% fetal calf serum.At 75% confluence, the cells were serum-deprived to inducedifferentiation (Halayko et al, 1999) and grown in serum-free basalmedia (Clonetics). Cells were studied between days 8 and 14 after serumstarvation. ASM phenotype was confirmed by positive staining for markersof smooth muscle cell differentiation including myosin kinase lightchain, and negative staining for factor VIII. ASM cells were used withinthe first 9 passages and perpetuated by trypsinizing cells forpropagating at a 1:4 dilution.

Madin Darby canine kidney (MDCK) cells transfected with the cDNA forhuman pIgR were used as positive control cells (MDCK-HpIgR, from Dr.Charlotte Kaetzel) for detection of pIgR, and the myelomonocytic cellline, U937 (ATCC), was used for its high level of endogenous expressionof Fc-alpha receptor.

Immunofluorescence Studies:

ASM are grown on collagen-coated coverslips in serum-containing growthmedium to 70% conflurence and then replaced with serum-free medium for 8to 14 days. After 16 hours treatment with pIgA, migA, rabbit anti-humansecretory component (SC; Dako) or media alone, the coverslips werewashed twice with ice cold PBS and fixed with 4% paraformaldehyde in PBSfor 20 min on ice. After washed three times in PBS, the coverslips wereblocked with 5% horse serum in PBS and 0.2% triton X-100 for 1 h at 37°C. Goat anti-alpha chain or rabbit anti-human SC at 1:100 dilution inblocking buffer were incubated with the coverslips for 1 h at 37° C.After washed three times in PBS and 0.05% triton X-100, the coverslipswere incubated with secondary antibody FITC-labeled donkey anti-goat ordonkey anti-rabbit at 1:200 dilution in blocking buffer for 45 min at37° C. The coverslips then were mounted onto slides with one drop ofmounting medium (Vector laboratories, Burlingame, Calif., USA) andfluorescence images were observed and captured with an Olympusfluorescence microscope with a digital camera. Live cell uptake of IgAwas determined by staining fixed cells with a goat polyclonal antibodyto the alpha chain of IgA 1:100 (Jackson Laboratories). FcαR stainingwas detected with a mouse monoclonal IgG1 antibody to the receptor 1:25(BD Pharmingen) and a FITC-conjugated donkey anti-mouse IgG antibody.

Protein and RNA Expression:

Western blotting. ASM cells were lysed in 0.5% SDS lysis buffer withprotease inhibitor (100 mM NaCl, 50 mM Tris [pH 8.1), 5 mM EDTA [pH8.0], 0.02% NaN₃, 0.5% SDS, 1 mM PMSF, 10 μg/ml aprotinin, 1 mM Na₃VO₄)and run on a 8% SDS-PAGE gel. Lysates of MDCK-HpIgR were used as apositive control. After transferring to nitrocellulose membrane(Bio-Rad) for 1.5 h at 100 voltage in transfer buffer (25 mM Tris, 192mM glycine, 20% methanol), Western blotting was performed using either arabbit anti-human secretory component antibody (Dako) or a goatanti-human secretory component antibody (Sigma). The anti-SC antibodywas detected with an HRP-labeled secondary goat antibody to rabbit IgG(Sigma) or donkey antibody to goat (Jackson Immunochemicals). Antibodybinding was detected on film (Amersham Pharmacia Biotech) using enhancedchemiluminescence (ECL) (Amersham-Pharmacia Biotech).

RT-PCR and DNA sequencing. For detecting mRNA expression of pIgR orFc-alpha receptor, total RNA was extracted from ASM, MDCK-HpIgR or U937cells with TRIzol reagent (Life Technologies). Total RNA extracted fromMDCK-HpIgR cells were used as a positive control for pIgR, while theU937 cells were used as the positive control for Fc-alpha receptor.Primers were designed to represent the cytoplasmic domain of pIgR andthe IgA binding domain of the Fc-alpha receptor (Table 1 shows thesequence of the sense and antisense primers). RT-PCR was performed usingoligo dT, mMLV reverse transcriptase and Taq DNA polymerase (LifeTechnologies). The RT-PCR products were run on a 1.2% agarose gel. Bandsfrom the ASM lanes were cut out and purified with QIAquick Gel Extractonkit (Qiagen) for DNA sequencing. DNA sequences were compared to those inthe Genebank of NCBI human genome database for identification.

Calcium Studies:

(1) Calcium measurements. ASM were grown in serum free media for 8 dayson 96-well fluorescence plates (Costar). After loading cells with Fura2-AM (Molecular Probe) for 3 h at 37° C., the ASM cells were treated intriplicate with either media alone, mIgA (12 μg/ml) or pIgA (12 μg/ml).Fluorescence was measured using a multiwell fluorescence plate reader(Molecular Dynamics) at excitation wavelengths of 340λ and 380λ andemission wavelengths at 500λ. Data was collected and calculated calciumconcentration using the Grynkiewicz formula: [Ca²⁺]_(i) (in nmol/L)=Kd[(R−R_(min))/(R_(max)−R)]×β factor. R is the ratio of fluorescence at340 and 380 nm, R_(max) and R_(min) are the ratios at 340 and 380 nm inthe presence of saturating Ca²⁺ and zero Ca²⁺, β factor is the ratio at380 nm in zero and saturating Ca²⁺, and Kd, the dissociation constant,is 224 nmol/L. At the end of the incubation, the conditions were washedoff, and digitonin and EGTA were added to calculate the maximum andminimum cytosolic calcium concentrations, respectively. The results ofthis experiment are shown in Table 1.

In another study, the effect of increasing concentrations of pIgA(0.12-12 μg/ml, in triplicate) was studied over 3 hours in serum-starvedASM grown in 96-well fluorescence plates for up to 10 days. Theseresults are shown in FIG. 16A. These methods were also used to study theeffect of a mouse monoclonal IgG1 antibody to J-chain 1 μg/ml(InnoGenex) on pIgA-induced changes in cytosolic calcium inserum-starved human ASM. These studies were undertaken to confirm thatthe effect of IgA is indeed mediated by ASM pIgR because the pIgRrequires the presence of J-chain on polymeric immunoglobulins forefficient binding and endocytosis (Johansen et al, 2001). For thisstudy, the ASM cells were treated in triplicate for 3 h at 37 C with (1)media alone, (2) mouse IgG1 anti-J-chain monoclonal antibody (InnoGenex)1 μg/ml, (3) pIgA 0.12 μg/ml, (4) pIgA 0.12 μg/ml and anti-J-chainantibody, (5) pIgA 1.2 μg/ml, and (6) pIgA 1.2 μg/ml and anti-J-chainantibody (data shown in FIG. 16B).

(2) Calcium imaging. Fluorescence spectrophotometric and imagingtechniques were used to study calcium signaling in Fura-2 loaded primaryhuman ASM grown to 70% confluence on collagen-coated coverslips andserum-starved for 10-14 days. Cells were loaded with Fura-2 (MolecularProbes) for 1 h at 37 C. Cells were monitored for X min to ensureimaging stability, prior to being perfused with either (1) HEPESbuffered KREBS solution containing 140 mM NaCl, 4.9 mM KCl, 1.4 mMKH₂PO₄, 1.2 mM MgCl₂, 11 mM Glucose, 25 mM HEPES, 2 mM CaCl₂ at pH 7.4and 37 C; (2) pIgA (purchased from Dr. Vaerman) 0.12-12 μg/ml diluted inthe KREBS buffer; or (3) mIgA (Pierce) 12 μg/ml in the KREBS buffer.Each cell was circled for automatic data processing of intracellularfree calcium concentration for each cell and images were stored. Datawas analyzed by Excel.

Results

Primary ASM Cell Cultures Express IgA Receptors by Immunofluorescence.

Human bronchial ASM cells from 3 different, non-asthmatic donors wereobtained from Biowhitaker/Clonetics. All cells were used within 9passages, and characterized by positive immunostaining with smoothmuscle-specific actin and myosin, but not with factor VIII whichrecognizes endothelial cells. Primary cell cultures of serum-starvedhuman bronchial ASM grown on coverslips were fixed and stained for pIgRwith antibody to human SC, the extracellular portion of pIgR. FIG. 8Aconfirms green staining for pIgR in one representative ASM sample fromthe 3 different human. In another experiment, serum-starved ASM wereincubated overnight with pIgA or mIgA, washed extensively, fixed withparaformaldehyde and then immuno-stained with antibodies to the alphachain of IgA. Only ASM incubated with pIgA immunostained for the alphachain (FIG. 8B, left panel), in contrast to ASM incubated with mIgAwhich does not bind pIgR (FIG. 8B, right panel). Live uptake experimentswere also performed on serum-starved ASM pre-incubated with media alone(negative control), rabbit antibody to SC (ligand for pIgR), or anirrelevant rabbit IgG (control antibody). The cells were washed, fixedand stained with a FITC-conjugated secondary antibody to rabbit IgG.FIG. 8C confirms live-uptake of the rabbit antibody to SC (right panel)in contrast to the irrelevant IgG (middle panel) or serum alone.

These results show that serum-starved ASM express the pIgR protein andthat it is capable of selectively binding its ligands (pIgA or rabbitantibody to SC) and not irrelevant Iigands (mIgA or Irrelevant rabbitIgG).

FIG. 9 shows that in contrast to serum-starved ASM, non-starving ASM doimmunostain for the FcαR (top panel). In addition, this FcαR expressionincreases after pre-incubation of live cells with either pIgA (middlepanel) or mIgA (lower panel), both of which can bind the FcαR. ThusFIGS. 8 and 9 confirm that ASM cells express both the pIgR and the FcαRprotein. Furthermore, ligands to FcαR upregulate its expression onserum-fed ASM.

Primary Cultures of ASM have IgA Receptors Detected by Western Blotting.

FIG. 10 shows a western blot that confirms the presence of pIgR in bothserum-starved and non-starving ASM. MDCK-HpIgR epithelial cells wereused as a positive control for pIgR protein expression. The results ofthese western blots confirmed the presence of a band consistent withpIgR (FIG. 10, upper panel). It is of interest that the pIgR band fromthe 12-day serum-starved ASM appeared as a singlet, in contrast to thepIgR band from transfected MDCK cells, which appears as a doublet due tophosphorylation of the receptor. Non-starving ASM expressed the doubletpIgR band, as do the 6-day starving cells (FIG. 10, upper panel).Stripping this blot and re-probing with 4G10 (UBI) monoclonal antibodyto phospho-tyrosine shows that the second band of pIgR is phosphorylatedin non-starving ASM and in transfected MDCK cells, as commonly foundwith many receptors (FIG. 10, lower panel).

FIG. 10 adds further evidence that ASM express pIgR protein and that itappears as a doublet due to phosphorylation when the cells are grownwith serum.

Primary Cultures of ASM have IgA Receptors Detected by RT-PCR

FIGS. 11A and 11B show that ASM have mRNA encoding the cytoplasmicdomain of pIgR by RT-PCR. MDCK-HpIgR cells were used as a positivecontrol for pIgR mRNA message. DNA sequencing of this band (339 bp)revealed sequence homology to the epithelial pIgR. These results confirmthat ASM express mRNA for pIgR.

FIG. 11A also shows that serum-starved ASM do not have mRNA encoding theIgA-binding domain of FcαR by RT-PCR (right lane of image on right). Incontrast, RT-PCR with serum-fed cells show that ASM do express FcαR(FIG. 11A, middle lane of image on right; and FIG. 11B). U937 cells wereused as a positive control for FcαR mRNA message. DNA sequencing of theRT-PCR product from non-starving ASM (241 bp) confirmed it is the FcαR.

These results show that ASM express mRNA for both pIgR and FcαR.Furthermore, serum-deprivation down-regulates mRNA expression for FcαRin ASM.

Polymeric IgA Increases Cytosolic Calcium Concentrations in ASM.

Incubation with pIgA significantly increased cytosolic calciumconcentrations in 8 day serum-starved ASM in contrast to mIgA or mediaalone (Table 2). The effect of pIgA was seen after 1 h, peaked at 1 h 15min and was sustained to 1.5 h, which was the duration of theexperiment. In another experiment, increasing concentrations of pIgAcaused an increased rise in cytosolic calcium in serum-starved ASM (FIG.16B). The peak effect was observed in these cells after 2 h and theexperiment was continued for a total of 3 h. These results confirm thatpIgA increases cytosolic calcium in a dose-response manner.

Fluorescence spectrophotometric and imaging techniques confirmed theseresults. Incubation of serum-starved ASM with pIgA significantlyincreased cytosolic calcium concentrations (FIG. 12A), in contrast toincubation with buffer alone which shows no change in calcium (FIG.12B). In FIGS. 12 and 13, each line represents a different cellmonitored for the duration of the experiment on the same day. Each graphrepresents one of 3-5 studies per condition. Of greatest interest, thepIgA-induced rise in cytosolic calcium concentrations occurredconsistently after about 1.5 h and manifested a sustained response withan oscillating pattern for the duration of the experiment.

Similar experiments were done after first testing the serum-starved ASMwith histamine, a known stimulus for ASM contraction andbronchoconstriction. After the histamine was washed off, a lowconcentration of pIgA (0.12 μg/ml) was added. After 1 h, cytosoliccalcium concentrations increased and rose 4-6 fold with repeat histaminestimulation after washing off pIgA (FIG. 13A). The time controlexperiment in FIG. 12B shows that pre-exposure to histamine sensitizesthe serum-starved ASM cells to a second histamine exposure 2 h laterwith less than a 2-fold increase in intracellular calcium concentration.When a similar experiment was done with a high dose of mIgA (12 μg/ml),re-exposure to histamine increased calcium levels less than 2-fold afterwashing of the mIgA (FIG. 13C), as occurred with buffer alone in FIG.13B. Finally, repeating this experiment with a high dose of pIgA (12μg/ml) after initial exposure to histamine and carbachol (anotherstimulus for ASM contraction and bronchoconstriction), caused a dramaticrise in cytosolic calcium concentrations beginning 50 minutes later andgoing off-scale at 66 minutes with over a 30-fold increase (FIG. 13D).

These results show a unique response of ASM to pIgA that has never beendescribed before. First, pIgA causes a consistent delayed rise incytosolic calcium concentrations. This delayed response provides awindow of opportunity to potentially alter cell signaling eventstriggered by the rise in cytosolic calcium concentrations. Second, pIgAcauses a sustained rise in calcium concentrations that oscillate. Thefrequency of these oscillations have been associated with increasing theactivity of the pro-inflammatory transcription factor, NFcκB (Hu et al,J. Biol. Chem. 274:33995-33998, 1999). Chronic airway inflammationcharacterizes asthma and has been associated with airway remodeling thatleads to irreversible changes. Third, histamine appears to sensitize ASMto pIgA. This particular finding potentially has major implications fortreatment of allergic asthmatics who develop upper airway infections.Fourth, the lack of response to mIgA and absence of FcαR in non-starvingASM indicate that pIgA mediates its effect via pIgR.

Finally, imaging of serum-starved ASM exposed to the highestconcentration of pIgA (12 μg/ml) during the Fura-2 calcium experimentsshows ASM contraction (FIG. 14). The cell that is circled in Frame 1contracts and disappears by Frame 11. Not all the cells respond at theexact same time or to the same degree. However, even the cell in thelower right hand corner also changes shape. These cells were grown oncollagen coverslips and were noticeably subconfluent, and may accountfor the differences in time and intensity of response. Cell contractionhas never been described before in response to pIgA. The fact that thishappens in serum-starved cells, again, implies that this pIgA effect ismediated via pIgR.

Example 4

The inventors have shown that anti-j chain antibody (commerciallyavailable mouse monoclonal IgG1) decreases pIgA-induced increase in.cytosolic Ca++ (pIgA 1.2 and 0.12 ug/ml) in Fura-2 loaded primarycultures of serum-starved human airway smooth muscles grown onfluorescence plates (results are shown in FIG. 16B). This data showsthat inhibiting the binding of pIgA to mesenchymal pIgR by, for example,targeting J-chain, modulates calcium responses.

Example 5

The inventors have studied the downstream effects of adding IgA toosteoarthritis and rheumatoid arthritis synovial fibroblasts. In oneexperiment, the inventors demonstrated that IgA increased NFkB DNAbinding in both RA and OA synovial fibroblasts (FIG. 4). In anotherexperiment, the inventors demonstrated that IgA increasedTNF-alpha-induced ICAM-1 expression by cell ELISA (see FIG. 5A). Inadditional experiments, the inventors demonstrated that IgA increasedICAM-1 expression in RA synovial fibroblasts by cell ELISA only in thepresence of an antibody to the alpha chain of IgA thereby forming animmune complex like a pseudo-rheumatoid factor (FIG. 5B).

The inventors also demonstrated that IgA increases TNFα-induced IL-8 andRANTES gene expression in synovial fibroblasts from patients, with bothrheumatoid arthritis (FIG. 6) and osteoarthritis (FIG. 7) by RNaseprotection assay.

Methods

ICAM-1 Protein Expression was Evaluated Using a Cell ELISA Method.

Primary cultures of RA and OA synovial fibroblasts were cultured on 96well plates until confluent. To study the effect of IgA and TNFα, cellswere incubated for 16 h with the following conditions in triplicate: (1)cell culture media alone (baseline ICAM-1 expression); (2) TNFα 10 ng/ml(positive control); (3) pIgA 1.2 μg/ml; (4) pIgA 1.2 μg/ml and TNFα 10ng/ml ; (5) mIgA 1.2 μg/ml; and (6) mIgA 1.2 μg/ml and TNFα 10 ng/ml. Tostudy the effect of an immune complex containing IgA, cells were treatedfor 24 h with (1) media alone (baseline expression); (2) goat polyclonalantibody to the alpha chain of IgA 2.4 μg/ml (Jackson Laboratories); (3)pIgA 1.2 μg/ml; and (4) pIgA mixed together with the antibody to IgA.Cells were washed and then fixed with 4% paraformaldehyde for 30 minuteson ice, and blocked with 2% bovine serum albumin in PBS at 37 C. Mouseanti-human ICAM-1 antibody (Sigma) diluted 1:500 in 1% BSA in PBS wasadded to the cells for 1 hour at 37° C., followed by goat anti-mouseHRP-conjugate IgG (1:5000) (Bio-Rad, Hercules Calif.) for 30 min at 37C. Plates were developed with the HRP substrate, TMB (Sigma) and thecolor change was stopped with 0.18M H₂SO₄. The absorbance at OD450 wasmeasured on a Spectramax 190 from Molecular Devices utilizing SOFTmaxPro software.

The effect of IgA on gene expression of inflammatory mediators wasdetermined by RNAse protection assay using a custom-made multi-probetemplate set purchased from BD Biosciences that included probes for IL-8and RANTES as well as housekeeping probes for L32 and GAPDH. Radioactivelabelling for the RNase protection assays was performed according to themanufacturer's directions. RA and OA synovial fibroblasts were incubatedfor 48 h with (1) cell culture media alone (baseline expression); (2)TNFα long/ml (positive control); (3) pIgA 1.2 μg/ml; (4) pIgA 1.2 μg/mland TNFα long/ml; (5) mIgA 1.2 μg/ml; and (6) mIgA 1.2 μg/ml and TNFαlong/ml. The autoradiograph was scanned with a phosphorimager to detectband intensities. Gene expression was quantitated as the ratio of theband intensity of the gene of interest to the intensity of ahousekeeping gene.

Example 6

The inventors have also shown that: (a) pIgA increases tension ex vivoin dog tracheal smooth muscle strips in contrast to no change with mIgA(does not bind pIgR) or with control buffer (FIG. 17) and (b)pre-incubation of these strips with pIgA enhances histamine-inducedincrease in tension (FIG. 18). These ex-vivo experiments with dog tissueconfirm the in vitro cytosolic calcium studies obtained with human ASM:not only does pIgA modulate cytosolic calcium and ASM contraction, butbinding to pIgR also. sensitizes the smooth muscle cells to non-specificcontractile agonists like histamine.

Tension experiments were done using canine tracheal smooth muscle stripsusing methodology similar to Antonissen et al (1980). Roughly equallengths of dog trachealis smooth muscle strips (˜1 by 10 mm) weremounted on a tissue tension apparatus that was calibrated so that 1 g oftension was reflected by two large boxes on the strip chart recorder'strace paper. The smooth muscle strips were perfused with warmedoxygenated KREBS-Henseleite buffer solution. Once the tissue wasconnected to the transducer hook, roughly 1 gm of passive tension wasadded to obtain the optimal alignment of actin and myosin within thesmooth muscle cells. The effect of different isoforms of IgA on tensionwas studied by incubating the strips for 16 hours at 37 C with (1)buffer alone (control); (2) human mIgA (4.6 mg/ml); (3) human pIgA (12mg/ml); and (4) human sIgA (4.6 mg/ml). Results were recorded as the netchange in tension (gm). FIG. 17 shows the change in tension obtained at5.5 h into the incubation period. This time-point reflects peak tensionchange.

For studies evaluating the effect of IgA on histamine-inducedcontraction, dog tracheal smooth muscle strips were mounted on thetissue tension apparatus and the tension was normalized in mN(=force/cm²). The responsiveness of the tissue samples to 80 mM KCl (totest for muscle viability and health) as well as to histamine 20 mM (anon-specific bronchoconstrictor) was determined. Each strip wasincubated for 4 h at 37 C with: (1) buffer alone (control); (2) humanmIgA (2.3 mg/ml); (3) human pIgA (6 mg/ml); or (4) human sIgA (2.3mg/ml). A lower dose of pIgA was used so as to cause a minimal change intissue tension. After washing off the test conditions, the ability ofeach strip to contract in response to histamine 20 mM was repeated.After washing again, the response to KCl was re-tested. The length andweight of each muscle strip was measured to normalize tensionmeasurements. FIG. 18 shows the percent change in histamine responsebefore the incubation period as compared to after 4 h treatment with theIgA isoforms.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. TABLE 1 Protein Primer sequences plgR: cytoplas- micdomain sense 5′ GAG CCC ACT CCC TGC TCT AAC 3′ antisense 5′ AGA AGA GGGGAA GGA CGG GAG 3′ FcαR: IgA bind- ing domain sense 5′ CCT CAG TCT GGGGCT TTC TTT 3′ antisense 5′ CTT GTT TGC GTC CAT GTG GTC 3′

TABLE 2 Cytosolic calcium concentration (nM) in serum-starved ASM at 100min after adding stimulus: Expt. control mlgA plgA*# 1 353.50 374.001556.00 2 501.46 1961.61  2481.45 3 640.25 853.74  556.78 4 537.25995.72 1465.87 5 445.43 588.41 1391.39 means 495.58 954.70 1490.30 S.E. 47.56 273.12  305.12T-TESTP = 0.08, mlgA versus control*P = 0.02 (P < 0.05), plgA versus control#P = 0.046 (P < 0.05), plgA versus mlgA

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1. A method for modulating the inflammatory response of a mesenchymalcell comprising administering an effective amount of an agent that canmodulate an IgA receptor on a mesenchymal cell to a cell or animal inneed thereof
 2. A method according to claim 1 to inhibit theinflammatory responses of a mesenchymal cell.
 3. A method according toclaim 2 to treat an inflammatory condition caused by an IgA binding toan IgA receptor on a mesenchymal cell.
 4. A method according to claim 3wherein the. inflammatory condition is arthritis.
 5. A method accordingto claim 4 wherein the arthritides is selected from rheumatoidarthritis, osteoarthritis or a spondyloarthropathy.
 6. A methodaccording to claim 3 wherein the inflammatory condition is selected fromCrohn's disease, ulcerative colitis, Behcet's disease, Sjogren's diseaseand a vasculitis.
 7. A method according to claim 3 wherein the conditionis asthma, chronic bronchitis, acute bronchitis, bronchialhyperreactivity, chronic obstructive pulmonary disease, emphysema,interstitial lung disease, bronchiectasis or airway remodelling.
 8. Amethod for modulating cytosolic calcium signalling in a mesenchymal cellcomprising administering an effective amount of an agent that canmodulate an IgA receptor on a mesenchymal cell to a cell or animal inneed thereof.
 9. A method according to claim 8 comprising administeringan effective amount of an IgA receptor antagonist to prevent or inhibitintracellular calcium signalling in a mesenchymal cell.
 10. A method forinhibiting the contraction of a mesenchymal cell comprisingadministering use of an effective amount of an IgA receptor antagonistto a cell or animal in need thereof.
 11. A method for inhibiting theproduction of inflammatory mediators or growth factors comprisingadministering an effective amount of an IgA receptor antagonist to acell or animal in need thereof.
 12. A method according to claim 1wherein the IgA receptor is pIgR or FcαR.
 13. A method according toclaim 2 wherein the IgA receptor antagonist inhibits the binding of pIgAto pIgR.
 14. A method according to claim 2 wherein the IgA receptorantagonist inhibits the binding of pIgA to FcαR.
 15. A method accordingto claim 2 wherein the IgA receptor antagonist is a scFv that binds pIgRor FcαR.
 16. A method according 1 wherein the mesenchymal cell is asmooth muscle cell.
 17. A method according to claim 16 wherein the cellis an airway smooth muscle cell.
 18. A method according to claim 1wherein the mesenchymal cell is a fibroblast.
 19. A method according toclaim 18 wherein the cell is a synovial fibroblast.
 20. A method ofdelivering a substance to a mesenchymal cell comprising administering toan animal or cell in need thereof an effective amount of a conjugatecomprising the substance coupled to an IgA receptor ligand.
 21. A methodaccording to claim 20 wherein the IgA receptor is pIgR or FcαR.
 22. Amethod according to claim 20 wherein the mesenchymal cell is afibroblast or smooth muscle cell.
 23. A method of detecting a conditionassociated with the activation of a mesenchymal IgA receptor on amesenchymal cell comprising assaying a tissue sample or cells from thesample for (a) a nucleic acid molecule encoding an IgA receptor or afragment thereof or (b) an IgA receptor or a fragment thereof.
 24. Amethod according to claim 23 wherein the IgA receptor is pIgR or FcαR.25. A method according to claim 23 wherein the condition is aninflammatory condition selected from arthritides, including rheumatoidarthritis, osteoarthritis, spondyloarthropathies, Crohn's disease,ulcerative colitis, Behcet's disease, Sjogren's disease andvasculitides.
 26. A method according to claim 23 wherein the conditionis asthma, chronic Dronchitis, acute bronchitis, bronchialhyperreactivity, chronic obstructive pulmonary disease, emphysema,interstitial lung disease, bronchiectasis or airway remodelling.
 27. Amethod of detecting IgA mediated bronchial hyperreactivity comprising:(a) administering an IgA receptor agonist to a patient; and (b)detecting bronchoconstriction in the patient wherein an increase inbronchoconstriction as compared to a control indicates that the patienthas IgA-mediated hyperreactivity.
 28. A method according to claim 27wherein bronchoconstriction is measured by listening for wheezing onchest auscultation.
 29. A method according to claim 27 whereinbronchoconstriction is measured by measuring a reduced forced expiratoryvolume at 1 second (FEV1).
 30. A method of detecting IgA-mediatedbronchial hyperreactivity comprising: (a) administering an IgA-receptoragonist to a patient and detecting bronchoconstriction; and (b)administering an IgA receptor agonist followed by a non-specificbronchoconstricting agent to the patient and detectingbronchoconstriction at a lower dose than when the nonspecific agent isadministered alone wherein bronchoconstriction in step (a) and/orbronchoconstriction induced at a lower dose of the nonspecific agentadministered without the IgA receptor agonist in step (b) would indicatethat the patient has IgA-mediated bronchial hyperreactivity.
 31. Amethod according to claim 30 wherein the non-specificbronchoconstricting agent is methacholine or histamine.
 32. A methodaccording to claim 30 wherein bronchoconstriction is detected with apulmonary function test such as clinical spirometry [=measurement ofFEV1 and FVC].